In this proposal, we plan to validate robust global biomarkers of inflammation in sickle cell disease (SCD). We will build on our previous pre-clinical studies in humanized SCD mice and clinical observations[1], and ongoing in-human research activities supported by an active FDA R01 (R01FD005341, PI: Manwani), titled: ?Phase 2 Trial of Gamunex (Intravenous Gammaglobulin) for Sickle Cell Acute Pain?, and Price Foundation award (PI: Manwani/Soma) titled ?Impact of Penicillin And Hydroxyurea On The Gut Microbiome And Associated Markers Of Disease Severity In Sickle Cell Anemia.? In both these ongoing in-human projects we are examining markers of neutrophil activation: b2 integrin conformational changes and the increased number of aged neutrophils in Sickle Cell Disease (SCD) patients. Quantitative changes of neutrophil activation markers are being assessed by flow cytometry (i) in response to a novel therapeutic agent, and in (ii) correlation with clinical and laboratory markers of disease severity. This proposal addresses the following high priority areas identified in OTA-19-007 for the Cure Sickle Cell Initiative: (1)Develop and validate assays to assess biomarkers of inflammation related to vascular injury in sickle cell disease (2)Validation of biomarker(s), using retrospective and/or prospective methods, with the intended use as primary and/or secondary outcome endpoints in evaluation of curative genetic strategies for sickle cell disease. In this project, we propose to utilize advanced analytical and clinical validation of markers of neutrophil activation to establish values in comparison to age and race-matched controls. We will also measure these markers in a cross section of patients that have been treated with curative therapies, such as stem cell transplantation or gene therapy. These studies will allow us to validate neutrophil activation as a potential biomarker of disease burden and response to therapy in SCD patients. Finally, we will establish precise and reproducible assays in a clinical hematology laboratory to allow potential for scale up and standardization of the assay across multiple clinical laboratories. The overall goal is to validate neutrophil activation assays as ?monitoring? and ?response? biomarkers as per BEST criteria (https://www.ncbi.nlm.nih.gov/books/NBK338448/). This will provide the necessary evidence to support their incorporation into prospective curative therapy trials/approaches as response biomarkers.